The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Enteroviruses belong to the Picornaviridae family of viruses. They are transmitted from person to person via direct contact with virus shed from the gastrointestinal or upper respiratory tract, affecting millions of people worldwide each year. They are often found in respiratory secretions, e.g., saliva, sputum, or nasal mucus, stool, and cerebrospinal fluid of an infected person. Enteroviral infection can result in a wide variety of symptoms ranging from mild respiratory illness (common cold), hand, foot and mouth disease, acute hemorrhagic conjunctivitis, aseptic meningitis, myocarditis, severe neonatal sepsis-like disease, and acute flaccid paralysis. Historically, poliomyelitis was the most significant disease caused by an enterovirus, poliovirus. However, there are additionally at least 62 non-polio enteroviruses that can cause disease in humans, including Coxsackie A viruses, Coxsackie B viruses, and echoviruses.
Parechovirus is a viral genus in the family Picornaviridae, a large family of non-enveloped, positive-sense, single-stranded RNA viruses that have an icosahedral capsid. The capsid is an arrangement of 60 protomers, each formed from 4 proteins (VP1 to VP4), and encloses the linear RNA genome. The parechovirus genus is composed of two species: Human parechovirus and Ljungan virus. Human parechoviruses are commonly spread and cause mild, gastrointestinal or respiratory illness, but have been implicated in cases of myocarditis and encephalitis. More than 95% of humans are infected by human parechovirus early in life, within two to five years of age.
Clinical detection of viruses is usually accomplished using any one of a variety of methods. For example, virus particles or nucleic acids may be isolated from a biological sample (e.g., cerebrospinal fluid, nasopharyngeal aspirates, throat swabs, blood fluids, fecal material, urine, etc.). A retrospective diagnosis may be made by serology. Complement Fixation Tests (CFT) are most widely used in this method, although hemagglutination inhibition (HAI) and enzyme immunoassays (EIA) may be used to give a type-specific diagnosis. For more rapid diagnosis, either antigen detection or RNA detection may be performed. However, screening for multiple antigens or nucleotide sequences may be necessary because of the large number of viruses in these families. In addition, nucleic acids usually must be extracted from a crude biological sample in order to accurately detect the presence of nucleic acids from microorganisms.
Given the high degree of complexity associated with preparing and processing viral nucleic acids from biological samples for detection, diagnosis, and/or quantitation in cases where rapid diagnosis is sought, there is a need for methods involving fewer steps, fewer technological requirements, and shorter durations. In addition, there exists a further need for methods that can detect and differentiate the multiple types of enterovirus and parechovirus from other viruses in human samples with the fewest number of steps and without the need to extract nucleic acids from the samples.